The malfunctioning of protein kinases (PKs) is the hallmark of numerous diseases. A large share of the oncogenes and proto-oncogenes involved in human cancers code for PKs. The enhanced activities of PKs are also implicated in many non-malignant diseases, such as benign prostate hyperplasia, familial adenomatosis, polyposis, neuro-fibromatosis, psoriasis, vascular smooth cell proliferation associated with atherosclerosis, pulmonary fibrosis, arthritis glomerulonephritis and post-surgical stenosis and restenosis. PKs are also implicated in inflammatory conditions and in the multiplication of viruses and parasites. PKs may also play a major role in the pathogenesis and development of neurodegenerative disorders.
For a general reference to PKs malfunctioning or disregulation see, for instance, Current Opinion in Chemical Biology 1999, 3, 459-465. In general, protein kinases are enzymes that mediate intracellular signalling by affecting a phosphoryl transfer from a nucleoside triphosphate to a protein acceptor that is involved in a signalling pathway. These phosphorylation events act as molecular on/off switches that can modulate or regulate the target protein biological function. These phosphorylation events are triggered in response to a variety of extracellular and other stimuli. Many diseases, such as those mentioned above (or hereinafter), are associated with abnormal cellular responses triggered by these types of protein kinase mediated events.
Initiation, progression and completion of the mammalian cell cycle are regulated by various cyclin-dependent kinase (CDK) complexes, which are critical for cell growth. CDK8 is a kinase that is involved in cell cycle control and also implicated in the regulation of transcription. CDK8 along with its closely related isoform or paralog CDK19 and together with its partner Cyclin C, MED12 and MED13 are components of multi-protein Mediator complex which couples action of transcription factors with the molecular machinery that carries out transcription, e.g. Cdk8 couple basal transcriptional machinery to sequence-specific transcription factors such as Notch, p53, β-catenin, and also repress the transcription of other genes (Rzymski, T. et al., Biochim. Biophys. Acta, Proteins and Proteomics (2015), e-publication ahead of print (doi:10.1016/j.bbapap.2015.05.011)). As Mediator independent roles CDK8 has been shown to act as part of a separate complex as a histone kinase (Knuesel M. T., et al., Mol Cell Biol. 2009, 29(3):650-61) phosphorylating H3 at S10, a mark associated with transcriptional activation of IER genes. CDk8 also interacts with acetyltransferase 2A (also known as GCN5L) and both proteins as a complex cooperatively phospho-acetylated histone H3 to generate the dual H3S10p/K14Acmark (Meyer, K. D., et al., EMBO Journal (2008), 27(10), 1447-1457).
Tumour development is associated with genetic alteration and deregulation of CDKs and their regulators, suggesting that inhibitors of CDKs may be useful as anti-cancer therapeutics.
Specifically, CDK8 is a serine-threonine protein kinase that is encoded by the CDK8 gene. It has been found that CDK8 is an oncogene that regulates β-catenin activity (see e.g. Nature (2008) vol. 455 (25) p 547-553 by Firestein et al and Cancer Research (2009); 69(20): p 7899-7901 by Firestein et al). CDK8 has been identified as a gene that both modulates β-catenin activity and is essential for colon cancer cell proliferation. The gene, which encodes a member of the mediator complex, is located at 13q12.13, which has been found to be a region of recurrent copy number gain in a substantial fraction (˜60%) of colon cancers. The expression of this gene is therefore implicated in the proliferation of colon cancer cells, and hence its suppression may inhibit such proliferation (Firestein et al. Nature (2008) vol. 455 (25) p 547-553; Firestein et al. Int. J. Cancer: 126, 2863-2873 (2010); Seo, J.-O., et al., Oncology Reports (2010), 24(1), 285-291. The expression of this gene has also been implicated in the proliferation of breast cancer (Xiao-Yu Li et al., Int. J. Clin. Exp. Pathol. 2014, 7(1):92-100; Xu D. et al., Nat. Commun. 2015, 6:6641), malignant melanoma (Kapoor A. et al. Nature 2010, 468, 1105), gastric cancer (Kim et al. Int. J. Oncol. 2011; 38(5):1375-83 2011; Song, Y.-Q., et al., Diagnostic Pathology (2014), 9, 64/1-164/6), ovarian cancer (Roninson et al. Proc. Natl. Acad. Sci. USA, 2012; 109(34):13799-804), and pancreatic cancer (Xu W. et al. Cancer Lett. 2015; 356(2 Pt B): 613-27). Porter D. C. et al. Proc. Natl. Acad. Sci. USA, 2012; 109(34):13799-804 have also reported that CDK8 expression correlates with poor survival in breast and ovary cancer. Given that CDK8 over-expression is characterised by high levels of CDK8 and β-catenin hyperactivity, CDK8 may activate β-catenin and other genes to drive colon cancer progression. Hence, inhibitors of CDK8 may be useful in the treatment of such cancers (by which we include reducing the progression thereof) given that they may inhibit the expression of genes important for oncogenic progression and controlled by CDK8 and/or they may regulate β-catenin activity.
CDK8 has been identified as a major kinase in the response to IFN signalling mediated STAT1-S727 phosphorylation (Bancerek J., et al., Immunity 2013 38(2):250-62). Moreover, it has been shown an inhibitory role of STAT1-S727 phosphorylation for NK cell cytotoxicity (Putz E. M., et al., Cell Rep. 2013 4(3):437-44), and knockdown of CDK8 verified its essential role for basal STAT1-S727 phosphorylation in NK cells and significantly enhanced cytotoxicity. This could be a novel immune cell-based strategy that in combination with other therapies could enhance clinical efficacy and outcome.
CDK8 is also implicated in the control of cell fate determination. Silencing of CDK8 using an inducible short hairpin strategy showed CDK8 expression is required for tumor growth in vivo, maintains tumors in an undifferentiated state, and regulates the expression of a subset of genes normally expressed in pluripotent embryonic stem cells in xenografts derived from cell lines that harbor copy number gain and overexpression of CDK8. CDK8 expression also plays a key role in regulating the pluripotent state in embryonic stem cells and MYC is an essential downstream target (Adler A. S., et al., Cancer Res. 2012 72(8):2129-39). Moreover, CDK8 expression is required to maintain embryonic stem cells in an undifferentiated state.
The pivotal role of CDKs in co-ordinating and driving the cell cycle in proliferating cells is proven, as are the biochemical pathways they are involved in. Specifically, as discussed above, it has been shown that CDK8 is linked to certain cancers. Given that there is a significant medical need for a targeted treatment of certain cancers, it is clearly of benefit to develop CDK8 inhibitors specifically.
Antimitotic treatments are also used to target cancer. Unfortunately, resistance to mitotic poisons is a recurrent problem in the clinic and new antimitotic therapies have demonstrated limited clinical responses probably due to the need for sustained exposures through a number of cell cycles or time in mitosis to elicit the maximum therapeutic response.
Haspin inhibitors could behave as strong mitotic cell death enhancers. Haspin inhibition of phosphorylation of histone H3 inhibits Survivin promoted chromosomal passenger complex (CPC) formation producing defects in chromosome segregation and cytokinesis. It is known that Survivin represses mitotic cell death (MCD). Therefore Haspin inhibition could be an indirect way to inhibit Survivin in mitosis.
Haspin (also known as germ cell-specific gene 2 protein/GSG2 or haploid germ cell-specific nuclear protein kinase) is a serine/threonine kinase (Tanaka H, et al., FEBS Lett. 1994, 355(1):4-10; Tanaka H et al., J Biol Chem. 1999, 274(24):17049-57; and Higgins J M, Gene 2001, 267(1):55-69). Haspin activity is restricted to mitosis. Haspin is most strongly expressed in testes, but also appears ubiquitously present in proliferating somatic cells (Higgins J M, Gene 2001, 267(1):55-69). Unlike mitotic kinases such as Aurora B and PLK1 that are degraded at the end of mitosis, human haspin is expressed at near-constant levels throughout the cell cycle (Dai J, et al., Genes Dev. 2005, 19(4):472-88).
In Huertas D, et al., Oncogene 2012, 31(11):1408-18, Haspin inhibitor CHR-6494 was shown to reduce H3T3ph in tumoral cells from colon, breast and cervix in a dose dependent manner and cause a mitotic catastrophe with a characteristic spindle and centrosome phenotype. The phosphorylation of H3T3 is crucial for the recruitment of Aurora-B to centromeres and its upstream activation (Kelly A. E., et al. Science (2010) 330(6001):235-9; Wang F., et al. Science (2010) 330(6001):231-5). H3T3ph is directly recognized by Survivin which is a member of the chromosomal passenger complex (CPC). This binding mediates recruitment of the CPC to chromosomes and activation of its kinase subunit Aurora B to ensure accurate cell division regulating kinetochore-microtubule attachments. It also establishes a positive feedback loop in which Aurora B further increases the kinase activity of Haspin (Wang F, et al. Curr. Biol. (2011) 21(12):1061-9). Modulation of phosphorylated H3T3 after synchronization or in normal growth conditions can be used to evaluate cellular Haspin kinase inhibition.
The identification of compounds that inhibit the activity of CDK8 and/or haspin represents a desirable drug design approach for the needed development of pharmacological agents for the treatment of diseases associated with CDK8 and/or Haspin.
For the treatment of cancer, targeted therapies are becoming more important. That is, therapy that has the effect of interfering with specific target molecules that are linked to tumour growth and/or carcinogenesis. Such therapy may be more effective than current treatments (e.g. chemotherapy) and less harmful to normal cells (e.g. because chemotherapy has the potential to kill normal cells as well as cancerous cells). This, and also the fact that targeted therapies may be selective (i.e. it may inhibit a certain targeted molecule more selectively as compared to other molecular targets, e.g. as described hereinafter), may have the benefit of reducing side effects and may also have the benefit that certain specific cancers can be treated (also selectively). The latter may in turn also reduce side effects.
Hence, it is a clear goal of current oncologists to develop targeted therapies (e.g. ones that are selective). In this respect, it should be pointed out that several different molecular targets may exist that are linked to certain diseases (e.g. cancer). However, one simply cannot predict if a therapy (e.g. a small molecule as a therapeutic) that interferes with or inhibits one target molecule could inhibit a different molecular target (be it one that will ultimately have the effect of treating the same disease or a different one).
Targeted therapies (such as CDK8 and/or haspin targeted therapy) could potentially have other advantages over current anti-cancer treatments, for instance because it may not directly interact with DNA (compared to certain known anti-tumour therapies) and should therefore reduce the risk of secondary tumour development.
Polycyclic compounds that are potentially useful as MAPKAP-K2 inhibitors are disclosed in Revesz L. et al. Bioorg. Med. Chem. Lett. 20 (2010) 4719-4723, and T.-J. Wang et al. Med. Chem. Res. (2013) 22:4818-4829. The compounds disclosed therein generally contain a tetracyclic core.
Compounds that are purportedly useful as CDK8 inhibitors are disclosed in WO 2014/154723. In the polycyclic compounds disclosed therein, the rings are not fused together but are coupled together via single bonds.
Pyrrolo-[2,3-f]-isoquinoline and dihydropyrroloisoquinoline compounds which are potentially useful as Cdc7 and AKT inhibitors are disclosed in WO 2008/065054. Tricyclic compounds which may be negative allosteric modulators of metabotropic receptors-subtype 5 are disclosed in WO 2010/049366. Polycyclic compounds which are potentially useful as PI3K inhibitors are disclosed in WO 2011/058149. None of these compounds are disclosed as being useful as CDK8 or haspin inhibitors, and the structures of these compounds differ in a number of ways from the compounds disclosed herein.
The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.